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Aryani and Denecke BMC Research Notes (2015) 8:164 DOI 10.1186/s13104-015-1114-z

RESEARCH ARTICLE Open Access In vitro application of : comparison of the effects on mRNA and miRNA stability Arian Aryani and Bernd Denecke*

Abstract Background: MicroRNA has become important in a wide range of research interests. Due to the increasing number of known , these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs. Methods: In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the -specific degradation grade depending on the RNA class was determined. Results: In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel . In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups. Conclusion: Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications. Keywords: mRNA, miRNA, Ribosomal RNA, Ribonuclease, RNA stability, RNA integrity

Background of their specific target [6]. The regulatory function of MicroRNAs (miRNAs) are small, non-coding which miRNA is differentiated into two distinct mechanisms: the are encoded within the . The , inhibition of target expression (i) by inducing the deg- pri-miRNA, contains one or more hairpin structures and is radation of the target mRNA [4] and/or (ii) by inhibition cleaved to pre-miRNA [1,2]. Upon export to the , of target gene [7]. Partial complementary base cleaves the pre-miRNA to mature miRNA, which pairing causes translational inhibition whereas perfect base usually has a length of about 22 [1,3,4]. pairing causes degradation of the complementary target Previous studies have suggested that mRNA stability is mRNAs. It has long been assumed that translational inhib- an important control point for regulation ition is the dominant mechanism, however, recent studies [5]. The mature miRNA plays a key role in the regulation have shown that the degradation of target mRNA plays the major role in the inhibition of expression [8]. A widespread interest has developed in small non-coding * Correspondence: [email protected] Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen), RWTH RNAs for cancer research and drug development. In Aachen University, Pauwelsstrasse 30, Aachen, Germany

© 2015 Aryani and Denecke; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aryani and Denecke BMC Research Notes (2015) 8:164 Page 2 of 9

particular, miRNAs appear to play a critical role in tumori- constituent of total RNA in mammalian cells [15]. For this genesis and are thus promising candidates as potential reason the quality of mRNA is generally extrapolated on therapeutic targets or as biomarkers [9,10]. Moreover, miR- the basis of the quality of ribosomal RNA, which can be NAs are able to regulate the expression of multiple genes analysed, for example, with microfluidic analysis (Agilent and hence affect disease pathways at multiple points [10]. 2100 Bioanalyzer). Therefore, miRNAs are promising tools in diagnostic and Representative electropherograms of our samples that therapeutic applications in a wide range of specializations, were analysed with the RNA6000 Nano labchip are including cancer research [11], cardiovascular disease [12], illustrated in Figure 1A. The electropherogram of the un- organ transplantation [13] and rheumatoid arthritis [14]. treated sample consists of four peaks: i) the loading Although miRNAs comprise an important class mole- marker at 25 nt, ii) the spiked tRNA at about 80 nt, iii) the cules used to uncover regulatory mechanisms in the , 18S ribosomal RNA and, iv) the 28S ribosomal RNA. The nearly nothing is known about their decay in comparison tRNA was added after the treatments in order to facilitate to mRNA under defined in vitro conditions. For this rea- the precipitation of RNA. It was clear that the 18S and son, we decided to enhance general knowledge about the 28S ribosomal RNAs were missing after NaOH treatment. stability of mRNA and miRNA in response to ribonucle- This was expected because NaOH, at the concentration ases commonly used in routine laboratory practice. We used, induces the of RNA in a manner that is were interested in finding out how a hypothetical contam- independent of size and sequence. Similar to NaOH, ination with ribonuclease might affect mRNA and miRNA RNase A-treated samples lacked both 18S and 28S peaks. stability. Furthermore, we investigated possible differences This indicated that both NaOH and RNase A treatments in the stability of different classes of RNA (i.e. miRNA, significantly affected ribosomal RNA. mRNA or ribosomal RNA). For this purpose, total RNA In the case of treatment with benzonase and RNase If, was treated in vitro with a range of ribonucleases under even though the ribosomal peaks were not detectable by identical experimental conditions and their effects were this method, the measured absorption was above the analysed by RT-qPCR. base line. This is caused by residues of ribosomal RNA. No specific changes in comparison to the untreated Results sample were observed in the electropherograms of the The integrity of ribosomal RNA is affected by treatment samples after treatment with RNase H, T or with specific ribonucleases Exonuclease T7. In contrast to mRNA, which represents only 0.5 to 3% of In order to determine the decay of ribosomal RNA the , ribosomal RNA is the most prevalent after the treatments with a higher sensitivity, a relative

Figure 1 The Stability of ribosomal RNA is affected in a treatment-dependent manner. (A) Representative electropherograms of untreated RNA (black)

and RNA after treatment with either Benzonase (dark orange), NaOH (pink), RNase A (red), RNase H (grey), RNase If (bright orange), Exonuclease T7 (dark blue) or Exonuclease T (bright blue). Electropherogram of RNA ladder, used for size calculation indicated in the abscissa as kilobases (kb), is illustrated in green. Arbitrary units for fluorescence are given in the ordinate. The untreated control sample represents four peaks: the loading marker at 25 nt, the spiked tRNA at about 80 nt, the 18S and the 28S ribosomal RNA. (B) Detection of 18S ribosomal RNA using RT-qPCR. Percentage of intact 18S ribosomal RNA after indicated treatments is illustrated for three independent experiments with mean and SD (in red). The relative percentage of intact RNA in comparison to the untreated RNA is given in the ordinate. The green dotted line represents the 100% intact 18S ribosomal RNA obtained from the untreated

RNA. Significances were calculatedbythetwotailedunpairedStudent’s t-test: * = p < 0.05; *** = p < 0.001. ExoT7 = Exonuclease T7;ExoT=ExonucleaseT. Aryani and Denecke BMC Research Notes (2015) 8:164 Page 3 of 9

measurement of 18S RNA was performed with RT- Nppb (Cq = 19.8 ± 0.8 cycles), Hprt (Cq = 24.7 ± 0.4 cy- qPCR (Figure 1B). The percentage of undigested 18S cles) and Polr2a (Cq = 26.9 ± 0.5 cycles). ribosomal RNA normalised to the untreated control is On the basis of our previous quantitative studies [19], presented in Figure 1B. It was observed that, similar to miR-1 and miR-208 (cardiac-specific expressed miRNAs) the results obtained by electrophoresis after treatment [20], sno202 (small nuclear RNA 202, often used as a with RNase A or NaOH, a significant reduction of ribo- normalization control) [21], and miR-501 [22] were in- somal RNA was detectable (a reduction of 77% and 66%, vestigated as indicators of miRNA stability. Figure 2B il- respectively). The observed difference in reduction be- lustrates the Cq values of the miRNA targets in the tween the two methods was probably caused by the untreated controls. Sno202 was more highly expressed higher sensitivity of the RT-qPCR. in heart tissue than the other miRNAs (Cq = 22.9 ± A similar effect could be observed after treatment with 0.5 cycles), followed by miR-1 (Cq = 23.3 ± 0.3 cycles) benzonase and RNase If. In contrast to a dramatic re- and miR-208 (Cq = 25.8 ± 0.2 cycles). For miR-501, the duction of ribosomal RNA detected by electrophoresis, lowest expression was detected (Cq =33.0 ± 0.3 cycles). the results obtained by RT-qPCR showed only a moder- To determine whether normalization to a reference gene ate reduction. In the case of benzonase, this reduction influenced the results, we normalized the mRNA values was significant (p = 0.027). For treatment with RNase If, using Polr2, and the miRNA values using sno202. As il- a statistically non-significant trend of reduction was lustrated in Figure 2C and D, the distribution of the observed. delta Cq values looked similar to the Cq values without The treatments with RNase H, T and Exo- normalization. The relatively high standard deviation for T7 showed no significant changes compared to mRNA seems to be caused by the different expression the untreated sample. This confirmed the result obtained levels between male and females. Such a gender distinc- by microfluidic analysis in a quantitative manner. tion was not visible for the analyzed miRNAs. In the case of RNase If treatment, comparison of sam- For both mRNA and miRNA, the selected targets ple electropherograms, which represented the 18S and cover a broad range of expression frequency (Cq 17.9 - 28S ribosomal RNAs (Figure 1A), with the results ob- 26.9 and Cq 22.9 - 33.0, respectively). This would ensure tained by 18S RT-qPCR (Figure 1B), revealed a discrep- the uncovering of potential treatment effects depending ancy between these two sets of results. The microfluidic on the frequency of a specific target RNA. However, a analysis revealed a partial degradation of 18S and 28S target RNA-dependent frequency was not observed. following treatment with RNase If , while the RT-qPCR Consequently, in order to compensate for the different demonstrated only a partial, non-significant digestion of target frequencies (i.e to have the result independent the 18S ribosomal RNA. from the original Cq value of each target), we normal- Taken together, these results indicate that, under ex- ized the results to the Cq value of the corresponding un- perimental conditions, RNase A, benzonase, NaOH and treated sample in our further analyses. RNase If treatments cause a total or partial decay of ribosomal RNA. Ribonucleases do not have the same effect on different classes of RNA Detection of mRNA and miRNA are represented by Next we considered whether the stability of mRNA and targets with different frequencies miRNA was the same after the different treatments. We Next we asked if the stability of ribosomal RNA to the measured the relative amount of intact mRNA/miRNA various treatments was representative of the stabilities of depending on the different treatments for the selected mRNA and/or miRNA to the same treatments. This targets. In Figure 3, the relative amount of the normal- knowledge is necessary to assess the relative influence of ised intact targets is shown as a percentage of untreated various treatments on different classes of RNA. For this control. For every treatment, the percentages of intact reason we performed RT-qPCR for different mRNA and targets for three independent experiments are repre- miRNA targets in the same samples as used for the riboso- sented for mRNA and miRNA. In addition, the sample mal RNA analyses. mean and standard deviation are shown. The results The mRNA stability was investigated by RT-qPCR for could be divided into five categories. expression of Hprt (normally used as a reference gene) [16], Polr2a (normally used as a reference gene) [17], i) The first category represented the significant decay Nppa (gene expressed in heart muscles) and Nppb (gene of mRNA and miRNA, including treatments with also expressed in heart muscles) [18]. First, we compared NaOH and RNase A. The RT-qPCR results for the the frequency of the targets analysed in the untreated mRNA targets suggested the total decay of mRNAs samples. As illustrated in Figure 2A, Nppa was most after the treatment with NaOH or RNase A. Similarly, highly expressed (Cq = 17.9 ± 0.4 cycles), followed by after the NaOH treatment, the average of undigested Aryani and Denecke BMC Research Notes (2015) 8:164 Page 4 of 9

Figure 2 Analysed targets represent a wide range of quantities. The quantification cycle (Cq), which is inversely proportional to the amount of target nucleic in the sample, was used to calculate the approximate amount of the targets. RT-qPCR analysis was performed with the untreated RNA isolated from three independent mice hearts (one male - grey, and two female - black). (A) Data-points and the mean of the Cq values of Nppa (cross), Nppb (downward-pointing triangle), Hprt (open circle) and Polr2a (hexagon) with the SD of three independent experiments are illustrated. (B) Data-points and the mean of the Cq values of Sno202 (closed circle), miR-1 (asterisk), miR-208 (rhombus) and miR-501 (upwards- pointing triangle) with the SD of three independent experiments are illustrated. (C) Data-points and the mean of the ΔCq values of Nppa (cross), Nppb (downward-pointing triangle), Hprt (open circle) and Polr2a (hexagon) with the SD of three independent experiments after normalization with the reference mRNA Polr2. (D) Data-points and the mean of the ΔCq values of Sno202 (upwards-pointing triangle), miR-1 (closed circle), miR-208 (asterisk) and miR-501 (rhombus) with the SD of three independent experiments after normalization with the reference small RNA sno202.

miRNAs was reduced by 48.5%. The reduction in a severe decay of miRNAs. These results for RNase A-treated samples was more prominent are consistent with those obtained by (i.e. 87.9%). Therefore, these treatments resulted microfluidic analysis and by RT-qPCR for the Aryani and Denecke BMC Research Notes (2015) 8:164 Page 5 of 9

could also be observed in the electropherogram of the microfluidic analysis. However, the partial digestion of 18S RNA represented by RT-qPCR was not significant (Figure 1B). iv) RNase H represented the fourth category, which caused no effect on mRNA or miRNA or ribosomal RNA. v) Benzonase represented the last category. After treatment with benzonase, a significant reduction of intact mRNAs was observed (20.4%). Contrary to this result, no reduction was seen for miRNA. The partial digestion of ribosomal RNA by benzonase as analysed by microfluidic analysis and 18S RT-qPCR was in line with the results of those for mRNA. Thus, benzonase precisely affected ribosomal RNA and mRNA, but had no effect on miRNA.

The results of our study indicate that, depending on the kind of treatment (i.e. ribonucleases), mRNA and miRNA demonstrate different stabilities. Notably, in com- parison to mRNA, miRNA frequently presented a higher level of stability. Taken together, our results indicate that NaOH and all analysed ribonucleases (except RNase H) affected mRNA and resulted in the decay of mRNA. On the one hand, benzonase, Exonuclease T7 and Exonuclease T significantly affected mRNA but had no significant effect Figure 3 mRNA and miRNA stabilities measured by RT-qPCR are differently influenced depending on the treatment. Percentage of on decay of miRNA. On the other hand, treatments with intact mRNA (blue) and miRNA (black) after indicated treatments are NaOH, RNase A and RNase If also affected miRNA. In illustrated for three independent experiments. Nppa, Nppb, Hprt, addition, after specific treatments of total RNA, microflui- and Polr2a were each analysed as targets representative for mRNA dic analyses revealed that ribosomal RNA was degraded and sno202, miR1, miR208, and miR501 each as targets for miRNA. whereas miRNA, as assessed by RT-qPCR, exhibited no The mean with the SD for all analysed targets of each RNA class is given in violet for mRNA and in red for miRNA. The green dotted degradation relative to the untreated sample. Hence, we line represents the 100% intact RNA obtained from the untreated conclude that there is not always a direct correlation be- RNA. The ordinate indicates the relative percentage of intact RNA tween the integrity of ribosomal RNA and that of mRNA in comparison to the untreated RNA. Significances were tested by or of miRNA. the two tailed unpaired Student’s t-test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. ExoT = Exonuclease T ; ExoT = Exonuclease T. 7 7 Discussion Significant amounts of miRNAs, which are remarkably ribosomal RNA, which also showed a significant stable, can be detected in all biological fluids. This has amount of decay. favoured the use of miRNAs as a new class of bio- ii) The second category consisted of Exonuclease T and markers. It is assumed that the high stability of miRNAs Exonuclease T7 treatments which resulted in the in biological fluids is based on the fact that they are pro- digestion of mRNAs but not of miRNAs. Although tected from endogenous RNase activity by being packed the reduction of undigested mRNA was only by inside exosomes or that they are protected via associ- about 2% in both cases, it was found to be ation with an AGO protein complex [23,24]. The pur- significant. As in the case of miRNA, ribosomal pose of the present study was to investigate the stability RNA did not show any changes in RNA integrity or of different classes of RNA (miRNA, mRNA, and rRNA) in 18S levels by RT-qPCR (Figure 1). against common sources of contamination in a molecu- iii)The third category was represented only by RNase lar biology laboratory. If. The amounts of intact mRNA and miRNA were In , the most common method used to as- both moderately, but significantly reduced (11.9% sess the integrity of RNA is the measurement of the 28S for mRNA and 6.6% for miRNA). This indicated to 18S ribosomal RNA where the ratio for intact RNA is that RNase If digested miRNA as well as mRNA, but 2:1. Previously, this assessment was done by running an in a moderate manner. Digestion of ribosomal RNA RNA aliquot on a denaturing agarose gel stained with Aryani and Denecke BMC Research Notes (2015) 8:164 Page 6 of 9

. Other stains such as biology laboratory. These ribonucleases are frequently SYBR Gold increase the sensitivity, also enabling the de- connected with a high possibility of reaction contamin- tection of lower amounts loaded on the gel. In the ation. To our knowledge, up to the time of this study, meantime, RNA integrity is assessed using microfluidic there is no information available regarding the in vitro analysis (e.g. Agilent 2100 Bioanalyzer, AdvanceCE™ Nu- effect of the analysed ribonucleases on classes of RNA. cleic Analyzer, or BioRad Experion). By contrast, In a previous study it was shown that, even in pre- absorbance or fluorescent dye-based procedures are use- sumed heat-degraded total RNA, the robust stability of ful for the quantification of RNA and, if necessary, for miRNA enabled its quantification. In contrast, the meas- the purity. These techniques are, however, unsuitable for urement was no longer possible for mRNA [27]. This the determination of RNA integrity. The assessment of study has also shown that the measured RNA integrity mRNA or miRNA integrity in total RNA, independent does not adversely affect the accuracy of RT-qPCR mea- of ribosomal RNA, is proving extremely difficult and surements of miRNA, although it significantly affects mostly includes RT-qPCR. Although there are also the measurement of mRNA [27]. Thus, degradation by microfluidic analysis assays for small RNAs (e.g. Agilent heat results in major effects on mRNA in comparison to small RNA assay) these assays are influenced by total miRNA, and miRNA represents a relatively stable form. RNA integrity [25]. For these reasons, microfluidic ana- Another study also indicated that miRNA possess dis- lysis of total RNA is widely used for the assessment of tinct stability. This stability depends on factors such as 7 total RNA integrity. The integrity of mRNA and miRNA on 3′ terminal or the specific sequences is normally extrapolated from the measured ribosomal within a miRNA that make the miRNA a target for ribo- integrity. nucleases. Also, some miRNAs are associated with com- Our results show that the degradation status of ribo- ponents of RISC such as Ago 2, which also influence the somal RNA is not, in all cases, applicable for the deg- miRNA stability [28]. However, in this study we were radation status of mRNA and miRNA. In addition, a more interested in comparing the stability of mRNA and class-dependent stability for mRNA, miRNA and ribo- miRNA after treatment with commonly used ribonucle- somalRNAwasdetectedinpresenceofspecificribo- ases, being the main source of contamination in routine nucleases. The class-dependent stability was supported laboratories. This important aspect by the observation that the analysed - RNA class- may affect the assessment of RNA quality and thus sub- specific - targets showed, more or less, the same stabil- sequent results of experiments. Precisely, we have shown ity. In general, miRNAs revealed more stability than that degradation by different ribonucleases is RNA class- mRNAs to exposure to ribonucleases and NaOH. This dependent. Our results also confirmed that RNA integ- indicates miRNAs to be good, candidate biomarkers. rity would not consistently affect the assessment of the Apart from the important regulatory effect of miRNA degradation of miRNA. on mRNA stability and/or translation [8], the dysregula- RNase A is an endoribonuclease that attacks single- tion of miRNAs can function as a biomarker for use in stranded RNA 3′ to residues and cleaves the cancer diagnosis and for many other diseases [10-14]. In linkage to the adjacent [29,30]. this context it is important to know if the observed dys- This RNase is one of the major contamination sources regulation is due to the regulation process or due to the in the molecular biology laboratory. NaOH also catalyses differential stabilities of mRNA and miRNA. the breaking of the phosphate backbone in RNA. As ex- Differential stabilities may have extensive consequences pected, both treatments (RNase A and NaOH) signifi- for analysis of the observed data. For instance, in the case cantly decayed all three analysed classes of RNA. of detection of increased levels of miRNA and a decreased Moreover, RNase If is one of the few known ribonucle- level of the corresponding target(s) mRNA, the conclusion ases which cleaves the between any would be that the putative down-regulation of mRNA is two nucleotides [31]. It is a single strand specific endori- due to the regulatory effect of miRNA. However, the lower bonuclease. It was reported that RNase If favours the stability of mRNA would not have been considered. Nor- degradation of small RNA but also de- malization to endogenous reference genes is a useful grades long RNA very slowly [32]. This earlier procedure to correct for sample-to-sample variations in finding would explain why all three examined classes of RT-qPCR efficiency and errors in sample quantification. RNA decayed, however, the observable decay for riboso- The expression is estimated relative to the levels of the mal RNA was not found to be significant. (invariant!) endogenous references [26]. However, this correct- RNase H cleaves RNA via ahydrolyticmechanismin ive action does not take effect between mRNA and miRNA, DNA/RNA duplexes [33]. This was confirmed in our exper- especially if these are degraded with different efficiencies. iments because no decay was observed in any class of RNA. Furthermore, we have performed the experiments with It was reported that Exonuclease T7 functions similar ribonuclesases that are commonly used in a molecular to RNase H on double-stranded DNA/RNA hybrids. It Aryani and Denecke BMC Research Notes (2015) 8:164 Page 7 of 9

specifically degrades the RNA region of poly(T)-poly(A) mRNA and ribosomal RNA). Consequently, the applica- hybrid polymers from 5′ terminus [34]. On the other tion of ribosomal RNA integrity as a proper representative hand, Exonuclease T is a single stranded RNA or DNA for mRNA and miRNA integrity should be treated with specific and removes nucleotides in the 3′ to 5′ caution, as it is not applicable in every case. direction. Exonuclease T also acts on single-stranded RNA in a sequence-specific manner. Both Exonucleases Conclusions do not decay ribosomal RNA and miRNA but they do In many studies, the putative down regulation of mRNA is decay mRNA. The small but significant decay of mRNA considered to be due to regulatory action of miRNAs. The by Exonuclease T might be due to sequence specificity different stabilities of RNA classes may cause false inter- of the , which has to be clarified in future stud- pretations of the observed data, especially when conta- ies. The small decay obtained for mRNA after treatment minations with RNases occur during the experiment. with Exonuclease T7 might have resulted from undefined Therefore it is important to consider and determine the cleavage, which is favoured for mRNA. stability of the respective classes of RNA under the condi- Benzonase degrades all forms of DNA and RNA and is tions of a given experiment. In our study we could demon- almost capable of cleavage at all positions along a nu- strate that after exposure to various RNases, miRNA is cleic acid chain. However, a sequence dependency was equally or even more stable than mRNA. This more demonstrated. A favourable digestion of DNA and RNA robust stability of miRNAs supports their potential and in a double-stranded G- and C-rich sequence has been further development as biomarkers in a wide range of ap- reported [29,35]. The reason for the observed missing plications. Due to the non-uniform stabilities of the vari- decay of miRNA could be explained by the small size of ous RNA classes under certain conditions, the application miRNA. Either small size RNAs, in general, do not act of ribosomal RNA as a representative measure of mRNA as a target for benzonase or the probability is very low or miRNA integrity should be reconsidered. that the target sequence is represented in the miRNA. In the present study we have used RNA from male Methods and female mouse hearts. This is important to show that Heart isolation and RNA extraction results are consistent across both genders. Also we have DBA/2 mice (43 days old) from which hearts were obtained generated the results in three independent experiments to isolate the RNA were maintained and sacrificed by cer- to prove the reproducibility of the results and to high- vical dislocation according to the German animal welfare light the prominent effect of the ribonucleases. However, law (TierSchG: § 7a, Absatz 2, Nummer 1 - 11038A4). this study can be expanded by providing other RNA Total RNA from the heart was isolated using peqGOLD samples or using other treatments. Genome-wide ana- RNAPure according to the manufacturer’s instructions lyses of the treatment-mediated effects on the different (PEQLAB Biotechnologie, Erlangen, Germany). classes of RNA are possible by using microarray technol- ogy. Using this method all targets would be amenable to RNases treatment assay support the observed RNA class-dependent stability and Each the equal amount of total RNA from an identical thus to generalize our findings. One limitation of our RNA pool was used for the different treatments (each study was to focus solely on heart tissue, although we 2.5 μg). In a volume of 100 μl total RNA treatment was have investigated not only heart-specific mRNAs and performed with indicated ribonucleases or NaOH (Table 1) miRNAs but also general expressed targets. at 37°C for 3 min in 1 × reaction buffer (1 mM DTT, Our study has confirmed the robust stability of miRNA 2 mM MgCl2, 2 mM CaCl2, 0.9% NaCl, 50 mM Tris, relative to mRNA. The observed stability is dependent on pH 8.0). The ribonuclease treatment reactions were the kind of treatment and on the kind of RNA (miRNA, stopped by mixing thoroughly after adding one volume of

Table 1 Ribonucleases/NaOH treatments with the concentration used in this study Treatment Units used per ml Supplier Enzyme commission number Literature RNase A 3.13 Applichem, Darmstadt, Germany EC3.1.27.5 [36,37] RNase H 12.50 Ambion, Kassel, Germany EC3.1.26.4 [38] Benzonase 8.33 Sigma, Steinheim, Germany EC3.4.23.1 [39] Exonuclease T 12.50 New England Biolabs, Frankfurt, Germany EC3.1.3.1 [40-42]

Exonuclease T7 12.50 New England Biolabs EC3.1.11.3 [34]

RNase If 12.50 New England Biolabs EC3.1.27.6 [31,32] NaOH 100 μmol Applichem Aryani and Denecke BMC Research Notes (2015) 8:164 Page 8 of 9

Roti®-/chloroform (Carl Roth, Karlsruhe, Germany). Table 2 TaqMan assays used in this study with the The NaOH treatment was stopped by adding the same efficiency rate for each detector molarity of acetic acid by vortexing. Detector Assay number Efficiency% To increase the efficiency of precipitation, a mix of 5 μg Hprt Mm01545399_m1 104.7 μ transfer RNA (Sigma) and 1.25 g Glycogen (Carl Roth) in Nppa Mm01255747-g1 106.4 a volume of 25 μl was added to the reaction suspension Nppb Mm00435304-g1 102.8 followed by intensively mixing for 15 sec. Then the sus- pension was transferred to a phase Lock Gel™ light 1.5 ml Polr2a Mm00839493_m1 94.7 tube (Eppendorf, Hamburg, Germany) and centrifuged for miR208 TM 511 89.5 15 min. Next, a volume of 90 μl of the upper phase was miR1 TM 2222 103.3 transferred to a new tube containing 9 μlof3Msodium sno202 TM 1232 97.9 acetate pH 4.8, mixed and incubated overnight at -20°C miR501 TM 1651 91.6 after adding 270 μl of 100% . The precipitate was TaqMan® Ribosomal RNA Control 4308329 centrifuged for 120 min, washed once with 100 μlof70% Reagents ethanol, and resuspended in 15 μl of ribonuclease-free water. All were performed at 44,000 × g and 4°C. Quantification of 18S ribosomal RNA was performed in RNA quantification was determined by optical density triplicates with the TaqMan® Ribosomal RNA control re- (Nanodrop ND-1000, PEQLAB Biotechnologie). For all agents () in accordance with the manu- analysed RNAs the observed ratio 260/280 was between facturer’s instructions on the ABI 7300 Real-Time PCR 1.8 and 2.0. RNA quality (integrity) was assessed using System at 50°C for 2 min, 95°C for 10 min, followed by microfluidic analysis (RNA 6000 Nano - Agilent 2100 40 cycles of 95°C for 15 sec and 60°C for 1 min. Assay effi- Bioanalyzer, both Agilent). ciencies of detectors were determined by serial dilutions within a 4-log range of the heart total RNA and calculated Reverse quantitative Chain with the formula 10-1/slope-1 (Slope = ΔCq/Δlog dilution) Reaction (RT-qPCR) [21] (Table 2). For mRNA analysis, cDNA was reverse transcribed from Since reference genes are also influenced by the 6 μl (40%) of resuspended RNA precipitate (see above) treatments, relative quantification was performed by using Superscript™ II , Oligo(dT) 12- normalization to the untreated control. For this purpose, 18 and random hexamer primers (all from Applied Biosys- the Cq value of 40 cycles was assumed to represent the tem), dNTP mix (Thermo scientific, Bonn, Germany) and total digestion of RNA (0% of RNA undigested) and the DTT (Invitrogen) according to manufacturer’s instruc- Cq value of the untreated control was assigned as not tions. To quantify the expression of digested (100% of RNA undigested). The undigested part phosphoribosyl (Hprt), natriuretic type after treatment was calculated using the following for- A(Nppa), natriuretic peptide type B (Nppb) and DNA- mula:x=100*(40-Cqtreat)/(40 - Cqunt); [x = percentage directed RNA polymerase II polypeptide A (Polr2A) real- of undigested after treatment, Cqtreat = Cq value of treated time PCRs using individual TaqMan® Gene Expression sample, Cqunt = Cq value of untreated sample]. Assays (Applied Biosystems) were performed in triplicates on an ABI 7300 Real-Time PCR System at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and Statistical analyses 60°C for 1 min. Subsequently, cDNA was mixed with The data were analysed with the Prism 5.0 software TaqMan® Gene Expression Master Mix (Applied Biosys- (©Graphpad Software Inc., USA). The significance was tems) and the appropriate TaqMan® Gene Expression As- calculated by the two-tailed unpaired Student’s t-test: *: says (Applied Biosystems) for the gene of interest (Table 2). p < 0.05; **: p < 0.01; ***: p < 0.001. SD = standard Quantification of selected miRNAs was performed using deviation. individual TaqMan® MicroRNA Assays according to the manufacturer’s instructions (Applied Biosystems). Shortly, Competing interests 6 μl (40%) of resuspended RNA precipitate (see above) The authors declare that they have no competing interests. were reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). miR-1, Authors’ contributions miR-208, miR-501 and snoRNA202 (Table 2) were quanti- BD designed the conception, hypotheses and delineation of the study. AA fied in triplicates on the ABI 7300 Real-Time PCR System performed the experiments. The above-mentioned authors conducted the required experiments, performed the acquisition of the data or analysed at 95°C for 10 min, followed by 40 cyles of 95°C for 15 sec such information. AA and BD prepared the manuscript and reviewed before and 60°C for 1 min. submission. Both authors read and approved the final manuscript. Aryani and Denecke BMC Research Notes (2015) 8:164 Page 9 of 9

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